Neurotransmitter and neuromodulator release takes place by exocytosis; the influx of calcium into the cell or nerve terminal triggers the fusion of the storage granule with the cell plasma membrane. The membrane fusion events can be modeled by studying the fusion of artifical or biological membranes with each other. A new multichannel, computer controlled stopped-flow rapid mixing spectrometer has been constructed to study the kinetics of these reactions. Our previous stopped-flow mixing studies have shown that the kinetics of both aggregation and fusion of small vesicular structures (artifical lipid vesicles, neurotransmitter storage granules, etc.) follow second order kinetics with fusion being aggregation rate limited. Our original stopped-flow assay for membrane fusion, based on resonance energy transfer between fluorescent phospholipids was subject to artifacts arising from the interactions of NBD-labelled phosphatidylethanolamine with the fusion catalysts such as Ca2+ interacting directly with the NBD probe and changing its quantum yield. We have developed a new assay, based on changes in the fluorescence of pyrene-labelled phosphatidylcholine. This assay examines the ratio of pyrene excimer/monomer fluorescence as a function of time after mixing. This ratio is independent of calcium concentration and is linearly dependent upon the distance between pyrene fluorophores, thus solving these problems and additionally allowing easy calculation of expected amplitude changes as the vesicles fuse together. This assay has been applied to the Mg-promoted fusion of artifical vesicles as well as protein-catalyzed fusion.